Estrogen receptors (ER) are closely involved in the processes which regulate growth and regression of breast cancer cells. It is believed that estrogens and their antagonists bind receptors and that the resultant complexes bind to specific nuclear acceptor sites and effect appropriate genes. It is the objective of this proposal to study in detail the nuclear loci of estrogen receptor action contrasting the effects of estradiol and antiestrogens on intranuclear receptor localization and on nuclear receptor turnover or processing with a view to demonstrating the site of divergence between estrogen and anti-estrogen action. These studies will proceed along three lines: (1) In acutely (5 min. to 5 hrs.) estradiol treated human breast cancer cells in tissue culture (MCF-7) we will trace the fate of the nuclear receptor by studying the roles of proteolysis, recompartmentalization and loss of hormone binding capacity. This will be done with parallel studies using routine receptor binding assays and with ER immunoassays. Monoclonal anti-ER antibodies will be produced by fusion of mouse myeloma cells to spleen cells from human ER immunized mice. (2) We will use actinomycin D and other inhibitors to probe the nature of ER interactions with chromatin using radioligand binding assays and immunoassays. (3) We will contrast these studies on estradiol with those on antiestrogen bound nuclear receptors to show whether antagonist properties result from aberrant nuclear receptor processing or binding to inappropriate chromatin sites.